All publications, references, patents, patent publications and patent applications cited herein are each hereby specifically incorporated by reference in its entirety.
RNA interference (RNAi) is a form of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) induces the enzymatic degradation of homologous messenger RNA (mRNA). When a long dsRNA enters a cell, an enzyme called Dicer binds and cleaves long, dsRNA. Cleavage by Dicer results in the production of a small interfering RNA (iRNA) that is generally 20-25 base pairs in length and has a 2-nucleotide-long 3′ overhang on each strand. Generically, an interfering RNA is also called an interfering nucleic acid (iNA), because non-RNA nucleotides can be incorporated into the construct. One of the two strands of each iNA, generally the antisense strand, is then incorporated into an RNA-induced silencing complex (RISC), and pairs with complementary sequences. RISC first mediates the unwinding of the iNA duplex. A single-stranded iNA that is coupled to RISC, then binds to a target mRNA in a sequence-specific manner. The binding mediates target mRNA cleavage by Slicer, an argonaute protein that is the catalytic component of RISC. The cleavage of the mRNA prevents translation from occurring, which prevents the ultimate expression of the gene from which the mRNA was transcribed.
As the fragments produced by Dicer are double-stranded, they could each in theory produce a functional iNA. The strand selected to be that with a less stable 5′ end.
RNA interference has a tremendous potential in medicinal therapeutics, such as in anti-viral, oncogenic and anti-inflammatory applications. The double-stranded iNA may be a long double-strand designed to be cleaved by Dicer, called Dicer substrate. Or the iNA may be short and designed to bypass Dicer serve directly as a RISC substrate. The dsRNAs are synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, RNA interference can cause a drastic decrease in the expression of a targeted gene.
Medicine
RNAi interference can be used to develop a whole new class of therapeutics. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful. Among the first applications to reach clinical trials were in the treatment of age-related macular degeneration, and respiratory syncytial virus. Other proposed clinical uses center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and co-receptors for HIV, the silencing of hepatitis A, hepatitis B and hepatitis C genes, silencing of influenza gene expression, and inhibition of measles viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease. RNA interference is also often seen as a promising way to treat cancer by silencing genes differentially up-regulated in tumor cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy.
Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for “off-target” effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed. A computational genomics study estimated that the error rate of off-target interactions is about 10%. In mammalian cells, however, the use of RNAi for targeted gene silencing has been limited due to nonspecific effects induced by long dsRNAs, which result in interferon response. Therefore, for applications in mammals, iNAs had to be designed to be less than 30 based pairs in length to prevent the PKR response.
However, in developing a therapeutic drug for a mammal, it would be desirable to create a long iNA for use in RNAi. An example of this is an iNA that contains multiple therapeutic targets, such as a sequence that anneals to an mRNA that produces a ligand and a sequence that anneals to the mRNA that produces the receptor of the ligand. However, such a long dsRNA containing two targets would induce the interferon response resulting in undesirable side effects to the patient.
Thus, there is a need to produce iNAs that can target more than one mRNA or more than one target or subsequence on a single mRNA.